Fig. 1

Experimental and data analysis pipeline of SNP-based RNA-seq analysis. a Time course of sample collection. Flowers of Col-0/SRK14 were emasculated at stage 12, 16 h - 20 h before pollination with compatible (C24) or incompatible (C24/SCR14) pollen grains, respectively. Stigmas were harvested (dashed line) 0, 10, 60 min after compatible (C0, C10, C60) or incompatible (I10, I60) pollination for RNA extraction. Typical scanning electron microscope images observed 60 min after pollination, for compatible reaction (Col-0 /SRK14 x C24) with hydrated round pollen grains and pollen tubes, and incompatible reaction (Col-0 /SRK14 x C24/SCR14) with dehydrated ellipsoidal pollen grains and no pollen tube. Scale bar = 20 μm. b We performed whole genome sequencing and detected variants using GATK (McKenna et al., 2010). SNPs and short indels between Col-0/SRK14 and C24 were identified (1.). Then, we produced new reference genomes for Col-0/SRK14 and C24 introducing the identified SNPs into TAIR10 Col-0 genome (2.). After deriving predicted mRNA from the new genomes, we performed RNA-sequencing and got sex-specific isoform abundance by using a statistic tool ASE-TIGAR (Nariai et al., 2016) (3.). Read normalization and differentially expressed gene analysis were performed using DESeq2 (Love et al., 2014). Sequence quality was checked by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). DNA and RNA reads were cleaned with custom Perl scripts